A New Anticoagulant−Antidote Pair:  Control of Thrombin Activity by Aptamers and Porphyrins

Abstract

Aptamers are nucleic acids generated by in vitro selection techniques that are able to specifically bind to ligands such as proteins. 1 A series of aptamers targeting blood clotting factors have been described that are highly effective in inhibiting blood coagulation and thus act as anticoagulants. 2 In applications, such as cardiopulmonary bypass surgery, it is crucial to reverse the anticoagulant effect. For this reason, anticoagulant/antidote effector pairs are required, and only one such pair, consisting of heparin as anticoagulant and protamine as antidote, is currently used in daily clinical practice. However, the use of heparin is associated with potential drawbacks including immune reactions. 3 Aptamers that interfere with the blood clotting cascade in turn offer the advantage that antisense molecules can be easily designed that revert the aptamer's action, acting as antidote. 3 Recently, this approach has been developed further by introducing caged thrombin aptamers that can be controlled using light excitation. 4 The distinct structural features of the thrombin-inhibiting aptamer containing a G-quadruplex open an alternative approach to finetune aptamer function. Quadruplex-binding compounds such as the cationic porphyrin TMPyP4 (meso-5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine) have attracted much attention since they bear the potential to interact with G-quadruplexes found in telomere ends of the chromosomes and promoters of proto-oncogenes, thereby often displaying anti-cancer activities. 5 Here, we have investigated if G-quadruplex-binding porphyrins are suited to control the anticoagulant activity of a G-quadruplex-containing aptamer that binds and inhibits human R-thrombin. We demonstrate that a cationic porphyrin, namely, TMPyP4, is able to bind to the aptamer and hence interferes with its active conformation. Consequently, TMPyP4 antagonizes the aptamer-mediated inhibition of blood clotting in a concentration-dependent manner, thus representing a true small molecule antidote of the anticoagulant DNA aptamer.Interactions of porphyrins with G-quadruplex structures are well characterized. 6 Cationic porphyrins such as TMPyP4 are known to bind to quadruplex structures such as the vertebrate telomeric sequence as well as G-rich promoter elements in proto-oncogenes such as c-myc. 7 Recently, we have engineered hammerhead ribozymes that are regulated by a TMPyP4-quadruplex interaction. 8 Nevertheless, so far, it has not been demonstrated that such interactions could be used to control the activity of functional aptamers containing G-quadruplex elements. In Figure 1A, the crystal structure of human R-thrombin in complex with the quadruplex-forming DNA aptamer inhibitor is shown. 9 Since the aptamer folds into an active G-quadruplex structure and thus allows specific binding to the blood clotting factor thrombin, interaction of G-quadruplex-binding porphyrins might interfere with the capability of the aptamer to bind to thrombin. In order to test this hypothesis, we have first characterized the in...

Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer

“…Many research groups recently demonstrated that, among the thrombin inhibitors, the aptamers TBA 15 , TBA 29 , and a series of related modified derivatives, were effectively controllable in their anticoagulation activity adopting various methodologies, such as the use of porphyrins, of complementary oligonucleotides or of external stimuli (light or radiofrequency irradiations). In particular, on the basis of the high affinity of porphyrins for G-quadruplexes, it was shown that a small cationic porphyrin can act as an antidote for TBA 15 , reverting the anticoagulant activity of the aptamer (Joachimi et al, 2007). The same result was obtained subsequently by studying hemin, an iron-binding porphyrin which can be an efficient antidote for a modified bifunctional TBA, by efficiently replacing thrombin in the TBA-thrombin complex (Fig.…”

Die chemische Biologie von Aptameren

“…[209] Das erste zu diesem Zweck eingesetzte Aptamer war ein Anti-Thrombin-GQuadruplex bildendes 15-meres Oligodesoxynucleotid. [218][219][220] Es wurden bereits klinische Studien der Phase I mit dem Aptamer, das unter dem Namen ARC183 oder HD1 während Aortokoronarer-Bypass-Operationen (CABG) einsetzbar ist, durchgeführt. Da zum Erzielen einer Wirkung jedoch die Verabreichung hoher Dosen erforderlich war, wurde die Entwicklung jedoch anschließend gestoppt.…”

Anticoagulant therapy during cardiopulmonary bypass

“…The development of anticoagulants for use in extracorporeal circuits covers a vast array of compounds, ranging from thrombin-directed aptamers [102], recombinant tick anticoagulant peptide [103,104], pentasaccharide [105,106], recombinant nematode anticoagulant [107], anti-TF antibodies [108] and active site inhibited factor VIIa [109] to recombinant tissue factor pathway inhibitors [110]. The unique conditions and local environment that define extracorporeal circuits create major challenges in pharmacotherapy application and investigation.…”